The long-term goal of this project is to understand the behavior of introduced transposable elements in transgenic mosquitoes that are vectors of human disease. It is the central proposition of the research described herein that the ability of a transposable element to be used both as an efficient genetic tool in the laboratory in mosquitoes, as well as an effective "gene driving mechanism" of beneficial effector genes in the field is determined by both how the transposable element functions, and how it interacts with the new host mosquito genome. This proposition will be examined using the Hermes transposable element, which is a member of the hAT family of transposable elements, and which is an effective transformation vector of the mosquito, Aedes aegypti. The Specific Aims of this proposal are thus: 1/ to purify and characterize the Hermes transposase, 2) to understand the structure: function relationships of the Hermes element, 3) to examine interactions of the Hermes transposase, and 4) to isolate hyperactive mutants of Hermes transposase and to test them for hyperactivity in mosquitoes. Purified biologically active Hermes transposase will be used to determine the optimal physical and biological parameters for Hermes element excision and transposition and for the role that host factors may play in Hermes element transposition in mosquitoes. Regions of the both the transposase and the element, such as the inverted terminal repeats and adjacent conserved motifs, that are required for transposition will be identified and, using mutagenesis, their precise roles in this process determined. The role of circular forms of the Hermes element, which we have identified to be present in transgenic insects containing autonomous forms of Hermes element will be investigated. Finally, both eukaryotic and prokaryotic based expression systems, both of which produce active Hermes transposase, will be used to identify hyperactive forms of the Hermes element system that will then be tested for elevated levels of activity in Ae. aegypti. To date we know little about how the transposable elements used to genetically transform mosquitoes either work or behave in mosquito genomes. This now constitutes the most significant bottleneck to the extension of transposable element-based technologies into medically important mosquito species. This technology will complement existing approaches to the control of mosquito-borne disease control and will provide new avenues with which to approach urgent problems arising from the increase in the geographical distribution of these devastating diseases which impact both human health and national economies. This new technology will enable mosquito genes required for pathogen transmission to be identified and isolated, will improve the efficiency with which transgenic mosquito strains can be identified, and will also provide important data concerning the likelihood that genes that prevent pathogen transmission can be spread through mosquito populations by transposable elements.